The mating response pathway regulating fusion of two haploid cells of the budding yeast S. cerevisiae or activated in response to exogenously added pheromones has been a prototypical signal transduction pathway, whose analysis led to multiple important insights into the structure and function of mitogen-activated protein kinase (MAPK) cascades. A vast collection of genetic and molecular biology tools have been developed that continue to make this pathway a very attractive system for developing a progressively refined understanding of how the signals from the cell microenvironment are converted into well defined changes in gene transcription and cell morphology. However, our understanding of this important pathway is far from complete, in large part due to the lack of experimental tools for analysis of the pathway activity in more physiological contexts of gradient sensing and chemotropism, and the sequential projection formation in saturating pheromone gradients (the default response), as opposed to the relatively short term responses to spatially homogeneous pheromone concentrations. In this application, we propose to study the mating pathway within the contexts of gradient sensing and default response phenotypes using novel experimental designs and microfabricated devices. We propose to investigate the role of various modifiers of pathway activity, with a particular focus on the scaffold protein and various negative regulators. We will also explore the mechanisms of cross-talk between the pheromone pathway and other MAPK pathways, also relying on the novel experimental technology. The results of these quantitative experimental analyses will be integrated into a comprehensive computational model of the MAPK pathways activated in budding yeast. Conservation of MAPK pathways across species and the particular importance of these pathways in various human pathologies make this research especially significant for our understanding of human health and disease. PUBLIC HEALTH RELEVANCE: This application is focused on the systems approach to signal transduction in one of the prototypical and most studied pathways, the mating pathway in yeast. In spite of its apparent simplicity, this pathway regulates a plethora of cell responses, from changes in cell morphology and directed cell growth, to periodic morphogenesis and expression of hundreds of genes. We propose using a set of novel approaches to investigate the way the pathway can control this diverse set of phenotypic responses and build a quantitative understanding of the pathway function. We anticipate that our findings will be informative about the function of other pathways in yeast and higher organisms.